Molecular modeling annual

, Volume 1, Issue 2, pp 54–67 | Cite as

On the Entropic and Hydrophobic Properties Involved in the Inhibitory Mechanism of Carboxypeptidase A by its Natural Inhibitor from Potato

  • Baldomero Oliva
  • Cristina Marino
  • Xavier Daura
  • Miguel A. Molina
  • Francese Canals
  • Francese X. Aviles
  • Enrique Querol
ORIGINAL PAPER

Abstract

The inhibition of carboxypeptidase A (CPA) by its natural inhibitor from potato (PCI) has been widely analysed with theoretical and experimental methods. Several mutants of PCI have been obtained in order to study the physico-chemical properties related to the inhibition. Point mutations were performed in the C-tail of PCI given its fundamental role in the inhibition. The inhibition constant and the dissociation free energy of the complexes PCI-CPA was experimentally obtained for each mutant. The mutants were divided in two sets, those where the mutation was intrinsically affecting the conformation of the PCI C-tail, and those where the mutation affected the interaction between PCI and CPA. The crystallographic structure of PCI, as found in its complex with bovine carboxypeptidase A, was used to model the structure of these mutants.

Two theoretical approaches were performed to explain both sets of experimental results:

1) study of the structural features of wt PCI and mutant forms by molecular dynamics (MD) simulation, and

2) modelling of the interaction of the C-tail of PCI with CPA.

The first approach provides an explanation of the observed behaviour of the mutants of PCI, if the hypothesis is made of a direct relationship between the entropy of inhibition and the mobility of the C-tail of PCI. For the second set of mutants, the experimentally measured dissociation energies for the complexes PCI- CPA can be related to the theoretically estimated exposure to the solvent of the side chain of the mutated residue in the complex. In the case of the double mutation G35P+P36G, the importance of the main chain hydrogen bond between Gly 35 and Ala26, anchoring the C-tail to the core of PCI, as predicted by the MD simulations, was also supported by the experimental result.

The agreement between the theoretical approaches and the experimental results shows the appropriateness of our hypotheses and also the relevance of such a combined effort of experimental and computational molecular biology in protein engineering.

Keywords: Potato Carboxypeptidase Inhibitor, Molecular Dynamics, Computer Simulations, Inhibitor Engineering, Inhibition Mechanism, Excluded Volume, Free Energy of the Inhibitor-Enzyme Complex 
Abbreviations: CPA = carboxypeptidase A (EC., 3.4.17.1); PCI = carboxypeptidase inhibitor from potato; wt = recombinant wild-type; re = recombinant; del = deletion 

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Copyright information

© Springer-Verlag Berlin Heidelberg 1995

Authors and Affiliations

  • Baldomero Oliva
    • 1
  • Cristina Marino
    • 1
  • Xavier Daura
    • 1
  • Miguel A. Molina
    • 2
  • Francese Canals
    • 1
  • Francese X. Aviles
    • 1
  • Enrique Querol
    • 1
  1. 1.Institut de Biologia Fonamental, Universitat Autonoma de Barcelona, Bellaterra, E-08193 Barcelona, Spain (baldo@luz.uab.es)ES
  2. 2.Unitat de Bioquimica i Biologia Molecular. Dpt. Biologia. Facultat de Ciencies Experimentals i de la Salut. Universitat de Girona. Girona 17071 SpainES

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